Genomic Measurement of Alternative Splicing

描述(申请人提供)：选择性剪接是控制哺乳动物基因表达的关键过程，也是蛋白质多样性的主要来源。剪接调控的错误与许多疾病过程有关，包括癌症和遗传性神经肌肉系统疾病。然而，控制剪接调控的细胞电路大多是未知的。在全基因组范围内测量剪接变化的新方法使发现选择性剪接的协调调控网络成为可能。阐明这种协调控制背后的调控事件对于理解外显子群在发育和疾病期间是如何控制的至关重要。该项目将支持布莱克、FU和阿瑞斯实验室通过先前的R24资助，继续开发和传播用于测量替代剪接的平行技术。在最初的项目阶段，开发了几种不同的方法。最值得注意的是，成功地设计、打印了两个剪接敏感的微阵列，一个用于小鼠，另一个用于人类细胞，每个芯片测量约1000个基因中约1300个可选剪接事件的剪接，并用于捕获和分析数据。这些阵列被应用于一系列不同的实验，并成功地揭示了几个在细胞分化和动态平衡中重要的协调剪接控制系统。我们建议继续这一富有成效的合作，以实现以下目标：(1)我们将继续将前一个资金时期产生的阵列和分析方法应用于剪接调控问题，并将其扩展到更多研究剪接的实验室；(2)我们将改进剪接敏感阵列的设计和分析，使其更全面、更可靠，以及更广泛应用；以及(3)我们将开发一种有前景的新方法，使用高密度测序方法进行全基因组剪接分析。该项目将把剪接调控的研究扩展到全基因组水平，允许将特定的剪接调控途径整合到我们对基因调控和基因组功能的理解中。公共卫生相关性许多人类疾病，包括癌症和神经肌肉系统的遗传性疾病，都是由基因功能改变引起的，这一过程被称为替代前mRNA剪接。尽管剪接的个体变化与特定的疾病有关，但人们还不太清楚剪接程序如何影响细胞的更大生物学，从而这些程序中的异常如何导致疾病。这个项目将扩展我们在全基因组范围内检查剪接调控方法的工作，这将使我们能够阐明疾病中这些更大的基因变化程序。

DESCRIPTION (provided by applicant): Alternative splicing is a key process in the control of mammalian gene expression and a major source of protein diversity. Errors in splicing regulation are implicated in many disease processes, including cancer and inherited disorders of the neuromuscular systems. However, the cellular circuits that control splicing regulation are mostly unknown. New methods that measure splicing changes on a genome-wide scale make possible the discovery of coordinately regulated networks of alternative splicing. The elucidation of the regulatory events underlying this coordinate control will be essential for understanding how groups of exons are controlled during development and disease. This project will support the continued development and dispersal of parallel technologies for measuring alternative splicing initiated by the Black, Fu and Ares labs through prior R24 funding. In the initial project period, several different approaches were developed. Most notably, two splicing- sensitive microarrays, one for mouse and one for human cells, each measuring splicing of about 1300 alternative splicing events in about 1000 genes, were successfully designed, printed and used to capture and analyze data. These arrays were applied to a diverse set of experiments and were successful in uncovering several systems of coordinate splicing control important in cellular differentiation and homeostasis. We propose to continue this productive collaboration with the following aims: (1) We will continue to apply the arrays and analysis methods produced during the previous funding period to questions of splicing regulation, and we will expand their use to additional laboratories studying splicing; (2) we will improve the design and analysis of splicing-sensitive arrays to make them more comprehensive, and reliable, as well as more widely available; and (3) we will develop a promising new approach to genome-wide splicing analysis using high density sequencing methods. This project will broaden the study of splicing regulation to the level of the whole genome, allowing the integration of specific splicing regulatory pathways into our understanding of gene regulation and genome function. PUBLIC HEALTH RELEVANCE Many human diseases, including both cancer and inherited diseases of the neuromuscular systems, are caused by alterations in gene function through a process called alternative pre-mRNA splicing. Although individual changes in splicing have been linked to particular disorders, it is not well understood how programs of splicing affect the larger biology of the cell, and hence how abnormalities in these programs lead to disease. This project will extend our work on methods for examining splicing regulation on a genome wide scale that will allow elucidation of these larger programs of genetic change in disease.